1. Field of the Invention
The present invention relates to instrument and method for tandem time-of-flight mass spectrometry used for quantitative analysis and simultaneous qualitative analysis of trace amounts of compounds and used for structural analysis of sample ions.
2. Description of Related Art
[Mass Spectrometers]
A mass spectrometer ionizes a sample in an ion source, separates the ions according to each value of m/z (mass-to-charge ratio) by the mass analyzer, and detects the separated ions by a detector. The result is shown in the form of a mass spectrum in which m/z value is plotted on the horizontal axis, while the relative amount is on the vertical axis. The m/z values and relative intensities of compounds contained in the sample are obtained. Qualitative and quantitative information of the sample can be derived. Various methods are available for ionization, mass separation, and ion detection. The present invention is especially closely associated with mass separation. Depending on different principles of mass separation, mass spectrometers are classified as a quadrupole mass spectrometer (QMS), an ion-trap mass spectrometer (ITMS), a magnetic sector mass spectrometer, time-of-flight mass spectrometer (TOFMS), and a Fourier transform ion cyclotron resonance mass spectrometer (FTICRMS).
[MS/MS Measurement and MS/MS Instrument]
In a mass spectrometer, ions created in the ion source are separated according to each value of m/z in the mass analyzer, and the separated ions are detected. The result is represented in the form of a mass spectrum in which m/z values of the ions and their relative amounts are graphically plotted. This measurement is hereinafter referred to as MS measurement in contrast with MS/MS measurement described later. In the MS/MS measurement, certain ions created in the ion source are selected by the first stage of mass spectrometer (abbreviated as MS1). The selected ions are referred to as precursor ions and are permitted to spontaneously fragment or are forced to be fragmented. The resulting product ions are mass-analyzed in the later-stage MS spectrometer (hereinafter abbreviated as MS2). An instrument enabling this MS/MS measurement is referred to as an MS/MS instrument (FIG. 1).
In MS/MS measurement, the m/z values of precursor ions, the m/z values of product ions produced in plural fragmentation pathways, and information about their relative intensities are obtained. Therefore, structural information about the precursor ions can be derived (FIG. 2). Variations of the aforementioned combination of two mass spectrometers are present as MS/MS instruments capable of MS/MS measurements. Furthermore, there are many fragmentation methods including collision induced dissociation (CID) using collision with gas, photodissociation, and electron capture or transfer. The instrument associated with the present invention is an MS/MS instrument including two TOF-MS instruments connected in tandem. A CID-based fragmentation means is interposed between the two TOF-MS instruments. Generally, this system is referred to as TOF/TOF.
Fragmentation information derived by an MS/MS instrument using a CID method is different depending on different collision energy, i.e., on different kinetic energy of ions entering the collision cell. In the case of the presently utilized MS/MS instruments, the energies are classified into low-energy CID of the order of tens of eV and high-energy CID of several kV to tens of kV. The difference is affected by the instrumental configuration. This is summarized in Table 1.
TABLE 1MS1MS2collision energyQMSQMSlowQMSTOFMSlowTOFMSTOFMShighmagnetic sectormagnetic sectorhighMSMSmagnetic sectorQMSlowMSion-trap MSion-trap MSlowion-trap MSTOFMSlowFTICR-MSFTICR-MSlow
An advantage of high-energy CID is that side-chain information may be obtained in fragmentation of peptides in which tens of amino acids are chained together. It is possible to discriminate between leucine and isoleucine having the same molecular weight.
[Time-of-flight Mass Spectrometer (TOFMS)]
TOFMS is a mass spectrometer for finding mass-to-charge ratios of ions by giving a constant amount of energy to the ions so that the ions are accelerated and made to travel and finding the mass-to-charge ratios from times in which the ions arrive at the detector. In TOFMS, ions are accelerated with a constant pulsed voltage of Va. At this time, from the law of conservation of energy, the velocity of each ion is given by
                                          mv            2                    2                =                  qeV          a                                    (        1        )                                v        =                                            2              ⁢              qev                        m                                              (        2        )            where m is the mass of the ion, q is the electric charge of the ion, and e is the elementary charge.
The ion reaches the detector rearwardly placed at a certain distance L in a flight time T.
                    T        =                              L            v                    =                      L            ⁢                                          m                                  2                  ⁢                  qeV                                                                                        (        3        )            
Eq. (3) indicates that the flight time T differs depending on the mass mn of the ion. An instrument for separating masses by utilizing this principle is a TOFMS. One example of a linear TOFMS is shown in. FIG. 3. Furthermore, reflectron TOF-MS instruments in which improvement of energy focusing and extension of flight distance are enabled by placing a reflectron field between an ion source and a detector have enjoyed wide acceptance. One example of reflectron TOFMS is shown in FIG. 4.
[Spiral Trajectory TOFMS]
The mass resolving power of a TOFMS instrument is defined by
                              mass          ⁢                                          ⁢          resolving          ⁢                                          ⁢          power                =                  T                      2            ⁢            Δ            ⁢                                                  ⁢            T                                              (        4        )            where T is the total flight time and ΔT is a peak width. That is, if the total flight time T can be prolonged while maintaining constant the peak width ΔT, the mass resolution can be improved. However, in the prior art linear TOFMS and reflectron TOFMS, prolongation of the total flight time T, i.e., increase of the total flight distance, will immediately result in an increase in size of the instrument.
A multi-turn time-of-flight mass spectrometer is an instrument developed to realize high-mass resolving power while avoiding bulkiness of the instrument (see, M. Toyoda, D. Okumura, M. Ishihara and I. Katakuse, J. Mass Spectrom., 2003, 38, pp. 1125-1142). This instrument uses four toroidal electric fields in each of which Matsuda plates are combined with a cylindrical electric field. Ions are made to make multiple turns in an 8-shaped orbit. Consequently, the total flight time T can be prolonged. This instrument succeeds in conserving spatial spread and time spread at the detection surface up to the first-order term.
However, the TOFMS in which ions are made to make multiple turns in a closed orbit suffers from an overtaking problem. That is, since the ions make multiple turns in a closed orbit, small m/z ions with large velocities overtake large m/z ions with smaller velocities. Therefore, the fundamental concept of TOFMS that lighter ions arrive at the detection surface earlier is invalidated.
A spiral ion trajectory TOFMS has been devised to solve this problem. The spiral trajectory TOFMS is characterized in that the starting and ending points of the closed trajectory are shifted with respect to the plane of the closed trajectory in an orthogonal direction. This is achieved by a method of entering ions obliquely from the beginning (JP-A-2000-243345), by a method of shifting the starting and ending points of the closed trajectory in an orthogonal direction using a deflector (JP-A-2003-86129), or by a method using laminated toroidal electric fields (JP-A-2006-12782).
A further TOFMS based on a similar concept has also been devised (WO/2005/001878). In this instrument, the trajectory of a multiple reflection TOFMS (U.K. Patent No. 2080021) in which overtaking occurs is zigzagged.
[MALDI Technique and Delayed Extraction]
A MALDI technique is a method consisting of preparing a matrix (such as liquid, crystalline compound, metal powder, or the like) having an absorption band in the wavelength of the used laser light, mixing and dissolving a sample in the matrix, solidifying it, and irradiating the matrix with the laser light to vaporize or ionize the sample. In a laser-induced ionization method typified by the MALDI technique, the initial energies produced during ion creation are distributed over a wide range. To converge the energies in time, delayed extraction is used in most cases. The delayed extraction consists of applying a pulsed voltage with a delay of hundreds of nsec from laser irradiation.
A conceptual diagram of a general MALDI ion source and delayed extraction is shown in FIG. 5. A sample is mixed and dissolved in a matrix (such as liquid, crystalline compound, metal powder, or the like). Then, the matrix is solidified and placed on a sample plate. A second lens, a second mirror, and a CCD camera are arranged to permit one to observe the state of the sample. Laser light is directed at the sample via a first lens and a first mirror to vaporize or ionize the sample. The created ions are accelerated by voltages applied to an intermediate electrode and to a base electrode, and are introduced into the mass analyzer.
A sequence of steps for measuring the flight time in a delayed extraction process is also shown in FIG. 5. First, an intermediate electrode and a sample plate are placed at the same potential Vs. Then, after a delay of hundreds of nsec from reception of a signal indicating laser excitation from the laser, the potential Vs at the intermediate electrode 1 is varied to V1 at high speed to create a potential gradient between the sample plate and the intermediate electrode. This accelerates the created ions. The starting time of measurement of flight time is synchronized with the rise time of the pulsed voltage.
[Orthogonal Acceleration TOFMS]
The MALDI technique has a very good affinity with TOFMS because ions are created in a pulsed manner. However, ionization methods for mass spectrometric analysis include numerous methods of continuously creating ions such as electron impact (EI), chemical ionization (CI), electrospray ionization (ESI), and atmospheric-pressure chemical ionization (APCI). Orthogonal acceleration TOFMS has been developed to combine these ionization methods with TOFMS.
A conceptual diagram of TOFMS using orthogonal acceleration is shown in FIG. 6. An ion beam is created from an ion source, which creates ions continuously, and continuously conveyed into an orthogonal accelerator with kinetic energies of tens of eV. In the orthogonal accelerator, a pulsed voltage of plus tens of kV is applied to accelerate the ions in a direction orthogonal to the direction in which the ions are conveyed from the ion source. After the application of the pulsed voltage, the time taken until the ions arrive at the detector differs according to different mass of ion and so mass separation is performed.
[TOF/TOF]
An MS/MS instrument in which two TOFMS instruments are connected in tandem is generally known as a TOF/TOF instrument and chiefly used in an instrument employing a MALDI ion source. A prior art TOF/TOF instrument is composed of a linear TOFMS and a reflectron TOFMS as shown in FIG. 7. An ion gate for selecting precursor ions is interposed between them. The focal point of the first TOFMS is disposed near the ion gate.
There are various kinds of ion gates. A typical ion gate is a parallel-plate type in which two electrodes are placed opposite to each other. Another typical ion gate is the Bradbury-Nielson type in which voltages with different polarities are alternately applied to plural wires. In addition, a method of enhancing the ion selectivity by arranging two ion gates along the flight axis is also proposed (JP-A-2005-302728).
Precursor ions are allowed to fragment spontaneously (i.e., post-source decay (PSD)) in some cases. In other cases, precursor ions are forced to fragment in a collision cell placed ahead of the reflectron field of the first or second TOFMS. The advantages and disadvantages of the MALDI-TOF/TOF mass spectrometer are next described.
Advantages
PRO: 1: It can efficiently measure, using MS/MS technology, samples ionized by a MALDI technique.
PRO: 2: It is one of a very few instruments capable of fragmenting ions with high collision energy (about 20 keV) (see Table 1).
Disadvantages
CON: 1: The precursor ion selectivity is low.
CON: 2: The mass resolution and the mass accuracy of the MS2 are low.
CON: 3: Since product ions produced by PSD fragmentation and product ions produced by CID fragmentation are mixed, the resulting spectrum is complicated and difficult to analyze.
CON: 4: It is possible to select only one precursor ion. This leads to wasteful consumption of the sample.
Some reports of methods of overcoming the CONs 1 and 2 have been made as described later. However, CONs 3 and 4 are the fundamental drawbacks with prior art TOF/TOF and, therefore, it is difficult to solve these drawbacks.
[Problem 1 with Prior Art]
A first problem with the prior art is that the precursor ion selectivity is low. The precursor ion selectivity is associated with the effective flight time of TOF1 and with the performance of the ion gate. Often, the first MS of the prior art TOF/TOF instrument is a linear TOFMS as mentioned previously. Therefore, the effective flight distance is about 0.5 m. It is necessary to take account of the performance of the ion gate from spatial and timewise viewpoints. FIGS. 8A and 8B show the differences of the times in which ions having m/z of 999, 1,000, and 1,001 and kinetic energies of 20.0 keV and 19.9 keV arrive at positions of 0.3 m ahead and behind from the TOF1 focal point for ions from the time in which ions having m/z of 1,000 and kinetic energy of 20.0 kV arrive at the same positions in a case where the effective flight time of TOF1 is set to 0.5 m. The focal point (0 m in the horizontal axis) is the focal point of TOF1. It can be seen that ions having the same m/z value but different kinetic energies arrive at the same time.
It can be seen from the figures that ions differing in m/z by one unit show no time difference at the positions of 0.1 m ahead and behind and thus overlap each other. That is, it is impossible to separate these ions however short the response time of the ion gate. Furthermore, at positions located within 0.1 mm ahead and behind from the focal point where there is no overlap, the time difference is about 5 ns per m/z. In addition, the spatial difference is only 0.5 mm. Consequently, the ions cannot be separated. As a result, there is the restriction that the ion gate must be placed close to the focal point of TOF1. Moreover, the precursor selectivity of TOF/TOF is approximately 2 units in the neighborhood of m/z.
[Problem 2 with Prior Art]
A second problem with the prior art is that the mass resolution and mass accuracy of MS2 are low. The reason why the mass resolution and mass accuracy of MS2 are low is closely related to the problem 1 and to high-energy CID that is an advantage of the TOF/TOF instrument.
The kinetic energy Upro of a product ion produced by collision-induced dissociation can be given byUpro=(m/Mpre)×Upre where Upre is the kinetic energy of the precursor ion, Mpre is the mass of the precursor ion, and m is the mass of the product ion. For example, where the accelerating voltage is 20 kV and the valence of the precursor ion is 1, kinetic energy Upre is 20 kV and so, in principle, product ions having kinetic energies of 0 to 20 keV are created by fragmentation.
In this way, some methods have been proposed to converge ions having a wide range of kinetic energies. In one method (U.S. Pat. No. 6,441,369), the distribution of kinetic energies is suppressed by deceleration, fragmentation, and acceleration. In another method (U.S. Pat. No. 6,300,627), the potential in a certain space is varied quickly after fragmentation, and then ions are again accelerated. In a further method (U.S. Pat. No. 4,625,112), an electric field gradient-type reflectron field is employed. In still another method (JP-A-2006-196216), reacceleration and an offset parabolic ion mirror (reflectron field formed by a linear electric field and a parabolic electric field) are combined. However, with these methods, it is difficult to converge all the ions having a very wide range of kinetic energies. Generally, MS/MS measurements result in poorer mass resolving power than MS measurements using reflectron TOFMS.
The resolving power is deteriorated due to the structure of TOF/TOF instrument. As described previously in connection with the prior art, TOFMS is based on the assumption that all ions are at the same position at some measurement start time except for the initial distribution. However, where TOFMS instruments are connected in tandem, not all ions are identical in initial position because ions with different m/z are separated by TOF1 and because plural ions having different m/z values are introduced into the second TOFMS due to poor precursor ion selectivity. Consequently, the mass resolution and mass accuracy of TOF2 are deteriorated.
[Problem 3 with Prior Art]
A third problem with the related art is that the results of MS/MS measurement are complex. The maximum advantage of a TOF/TOF instrument is that it is one of a few instruments capable of high-energy CID. However, it is known that in the MALDI technology, a PSD (post-source decay) takes place generally. The fragment pathways of a PSD process are close to the pathways of low-energy CID. Furthermore, in the prior art TOF/TOF instrument, TOF1 is a linear TOFMS and so it is impossible to separate PSD ions. Therefore, fragmentations due to CID and PSD are simultaneously reflected in MS/MS measurement results. As a result, as pointed out in Problem 2, the MS/MS spectrum becomes very complex because of low resolution of MS2. In consequence, it is difficult to analyze the spectrum.
[Problem 4 with Prior Art]
A fourth problem with the prior art is that it is possible to measure the fragment pathway from only one precursor ion during one MS/MS measurement. Table 2 shows the results of calculations of the relationship between the mass of the first precursor ions and the mass of a precursor ion capable of being selected next in a case where plural precursor ions are selected in MS/MS measurements using a TOF/TOF instrument in which a prior art linear TOFMS and reflectron-type TOFMS are combined. L1 is the effective flight distance of the first (linear) TOFMS. L2 is the effective flight distance of the first (reflectron type) TOFMS. In the computation, L1/L2 was set to 0.5
TABLE 2mass of precursor ionmass of precursor ion capable of being selectedselected firstnext; in a case where L1/L2 = 0.55004,5006005,4007006,3008007,2009008,1001,0009,0001,1009,9001,20010,8001,30011,7001,40012,6001,50013,500
As can be seen from Table 2, the difference in mass between the first selected precursor ion and the next selected precursor ion is great. It has been effectively impossible to select plural precursor ions in one measurement. That is, since all the ions excluding the selected precursor ions are eliminated, the sample is consumed wastefully.